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Cold-resistant gene CsLAC18 of sweet oranges and application of cold-resistant gene CsLAC18

A technology of gene and sweet orange, which is applied in the field of separation in is), can solve the problem of unclear effect and achieve the effect of improving cold resistance

Active Publication Date: 2020-11-20
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In summary, laccase plays an important regulatory role in plant stress response, but its role in low temperature stress is still unclear

Method used

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  • Cold-resistant gene CsLAC18 of sweet oranges and application of cold-resistant gene CsLAC18
  • Cold-resistant gene CsLAC18 of sweet oranges and application of cold-resistant gene CsLAC18
  • Cold-resistant gene CsLAC18 of sweet oranges and application of cold-resistant gene CsLAC18

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Sweet orange laccase gene CsLAC18 Cloning of full-length cDNA

[0033] In this example, the CDS sequence was obtained from the sweet orange genome database (http: / / citrus.hzau.edu.cn / orange / ), and primers were designed in the 5' non-coding region and 3' non-coding region of the sequence, wherein Forward primer: CsLAC18 forward:

[0034] 5'-ATGGGAGCTTCTCTTCTTCGATC-3';

[0035] Reverse primer: CsLAC18 reverse :

[0036] 5'-TCAGCACTGAGGAAGATCTG-3'. Then, using sweet orange cDNA as a template, its full-length was amplified with McLab high-fidelity enzyme (purchased from Beijing Qingke Biotechnology Co., Ltd.).

[0037] The research material sweet orange was planted in the Plant Culture Room of the College of Horticulture and Plant Protection, Yangzhou University, and its seedling age was 60 days. The robust sweet orange seedlings were selected, 0.1 g leaf samples were randomly weighed, and quickly frozen with liquid nitrogen. Sweet orange RNA was extracted ...

Embodiment 2

[0052] Example 2 Sweet orange laccase gene CsLAC18 subcellular localization of

[0053] according to CsLAC18 The nucleotide sequence of the gene and the p101-YFP vector map (the vector p101-YFP was purchased from Guangzhou Huijun Biological Co., Ltd.), and the EcoRI and BamHI restriction sites were added before and after the gene sequence. The sequence of the restriction site is as follows:

[0054] EcoRI: GAATTC

[0055] BamHI: GGATCC.

[0056]The CloneExpress® II One Step Cloning Kit (Vazyme, China) was used to construct the vector in one step. For the ligation method, please refer to the kit instruction manual. Phanta® Max Super-Fidelity DNA polymerase high-fidelity enzyme (Vazyme, China) was used for amplification. The target gene extraction plasmid with correct sequencing results was used as a template, and primers added with restriction sites were used for amplification. The PCR amplification procedure was as follows: Pre-denaturation at 98°C for 3 min; denaturati...

Embodiment 3

[0069] Example 3 Sweet orange laccase gene CsLAC18 Application in Improving Cold Resistance of Tobacco

[0070] 1. Plant Transformation Vector Construction

[0071] according to CsLAC18 The nucleotide sequence of the gene and the plant binary expression vector pBI121 (purchased from Beijing Qingke Biotechnology Co., Ltd.), design the primer CsLAC18-pBI121-F / R and insert the full-length CsLAC18 gene into the Xba on the pBI121 vector Between I and Sma I restriction sites. Using sweet orange cDNA as a template, the primers were designed as follows:

[0072] CsLAC18-pBI121-F:

[0073] 5'- TCTAGA ATGGGAGCTTCTCTTCTTCGATC-3'

[0074] CsLAC18-pBI121-R:

[0075] 5'- CCCGGG TCAGCACTGAGGAAGATCTG-3'.

[0076] The amplified fragments were recovered, and the plasmid pBI121 was double-digested with restriction endonucleases Xba I and Sma I, electrophoresed and recovered after digestion, and then the vector was constructed by one-step method, and the amplified fragment was connec...

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Abstract

The invention provides a cold-resistant functional gene CsLac18 separated and cloned from sweet oranges; the nucleotide sequence of the cold-resistant functional gene CsLac18 is shown as SEQ ID NO.1,and the corresponding amino acid sequence of the cold-resistant functional gene CsLac18 is shown as SEQ ID NO.2. The gene CsLac18 is introduced into tobacco and fructus aurantii for functional verification, and it is found that the cold resistance of the obtained transgenic overexpression plant is obviously improved, and the cold resistance of the transgenic silent expression plant is obviously reduced. Important gene resources are provided for plant cold-resistant gene engineering, and the cold resistance of plants can be improved.

Description

technical field [0001] The present invention relates to a kind of sweet orange ( Citrus sinensis A low-temperature-induced laccase gene isolated and cloned from CsLAC18 , the gene has the function of cold resistance, and also involves a sweet orange cold resistance transporter gene CsLAC18 The application in improving the cold resistance of plants is to transform the above-mentioned genes into tobacco and trifoliate orange, and the cold resistance ability of the obtained transgene overexpression plants is obviously improved, while the cold resistance ability of the transgene silent expression plants is obviously reduced, which belongs to the technical field of plant genetic engineering. Background technique [0002] As the largest category of fruit in my country and even in the world, citrus is also the fruit tree horticultural crop with the widest cultivation area, the largest product output and the highest economic status in southern my country. my country's citrus ind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/11C12N15/82A01H5/00A01H6/82
CPCC12N9/0061C12Y110/03002C12N15/8273
Inventor 徐小勇张越良张亦武周一鹏孔维文
Owner YANGZHOU UNIV
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